1. Field of the Invention
The invention relates generally to cancer markers and more specifically to a method of evaluating gene methylation within a sample, referred to herein as quantitative multiplex methylation-specific PCR (QM-MSP), in order to detect conditions associated with methylation status of genes.
2. Background Information
Epigenetic alterations including hypermethylation of gene promoters are proving to be consistent and early events in neoplastic progression (Hanahan D. and Weinberg R. A. Cell (2000) 100:57-70; Wamecke P. M. and Bestor T. H. Cum Opin Oncol (2000) 12:68-73; Yang X. et al. Endocr Relat Cancer (2001) 8:115-127; and Widschwendter M. and Jones P. A. Oncogene (2002) 211:5462-5482). Such alterations are thought to contribute to the neoplastic process by transcriptional silencing of tumor suppressor gene expression, and by increasing the rate of genetic mutation (Wajed S. A. et al. Ann Surg (2001) 234:10-20 and Jones P. A. and Baylin S. B. Nat Rev Genet (2002) 3: 415-428). DNA methylation is reversible, since it does not alter the DNA sequence; however, it is heritable from cell to cell. Methylated genes can serve as biomarkers for early detection of cancer for risk assessment and for predicting response to therapy.
Widschwendter and Jones (supra) reviewed over 40 genes whose expression is lost in breast cancer due to promoter hypermethylation, and others have studied hypermethylation of genes including NES-1 (Goyal J. et al. Cancer Res (1998) 58:4782-4786; Dhar S. et al. Clin Cancer Res (2001) 7:3393-3398; Li B. et al. Cancer Res (2001) 61:8014-8021; and Yunes M. J. et al. Int J Radiat Oncol Biol Phys (2003) 56:653-657) APC (Kashiwaba M. et al. J Cancer Res Clin Oncol (1994) 120:727-731; Virmani A. K. et al. Clin Cancer Res (2001) 7:1998-2004; and Sarrio D. et al. Int J Cancer (2003) 106:208-215), Cyclin D2 (Evron E. et al. Cancer Res (2001) 61:2782-2787 and Lehmann U. et al. Am J Pathol (2002) 160:605-612), RARB (Widschwendter M. et al. J Natl Cancer Inst (2000) 92:826-832; Yan L. et al. J Mammary Gland Biol Neoplasia (2001) 6:183-192; and Sirchia S. M. et al. Cancer Res (2002) 62: 2455-2461), TWIST (Evron E. et al. Lancet (2001) 357:1335-1336), RASSF1A (Lehmann U. et al. Cancer Res (2001) 61:8014-8021; Burbee D. G. et al. J Natl Cancer Inst (2001) 93:691-699; and Dammann R. et al. Cancer Res (2001) 61:3105-3109), and HIN1 (Krop I. E. et al. Proc Natl Acad Sci USA (2001) 98:9796-9801) in tissue, blood and ductal fluids. Since methylation changes often appear early in disease, detection of hypermethylated genes could identify tissues derived from subjects with increased risk. Furthermore, the reversible nature of methylation offers the potential to revert aspects of the cancer phenotype with the appropriate therapy (Fackler M. J. et al. J Mammary Gland Biol Neoplasia (2003) 8:75-89).
Tumor DNA can be found in various body fluids and these fluids can potentially serve as diagnostic material. Evaluation of tumor DNA in these fluids requires methods that are specific as well as sensitive. For instance, a PCR-base technique called methylation-specific PCR (MSP) is reported to detect one copy of methylated genomic DNA in one-thousand unmethylated copies of genomic DNA (Herman J. G. et al. Proc Natl Acad Sci USA (1996) 93:9821-9826). This approach has been modified in order to co-amplify several genes simultaneously in a nested or multiplex MSP assay (Palmisano W. A. et al. Cancer Res (2000) 60:5954-5958; Buller A. et al. Mol Diagn (2000) 5:239-243; and Brock M. V. et al. Clin Cancer Res (2003) 9:2912-2919). The read out is gel-based and qualitative (“all or nothing”). This method has been used to establish the frequency of gene promoter hypermethylation among subjects with bronchial and esophageal carcinoma. However, the method cannot quantitatively measure the levels of gene methylation.
Quantitative real time PCR (Q-PCR) allows a highly sensitive quantification of transcriptional levels or levels of the DNA of the gene of interest in a few hours with minimal handling the samples (Heid C. A. et al. Genome Res (1996) 6:986-994 and Gibson U. E. et al. Genome Res (1996) 6:995-1001). cDNA or genomic copies of the gene of interest are quantitated by detecting PCR products as they accumulate using an optically detectable polynucleotide probe. This technique is widely used.
Quantitative MSP (Q-MSP) allows highly sensitive detection of gene promoter methylation levels by real time PCR with methylation-specific primers probes (Lo Y. M. et al. Cancer Res (1999) 59:3899-3903; Trinh B. N. Methods (2001) 25:456-462; Wong I. H. et al. Clin Cancer Res (2003) 9:1047-1052).
The advantage of fluorogenic probes over DNA binding dyes (e.g. syber green for real time PCR) is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. However, Q-MSP analysis of multiple genes requires additional quantities of template DNA. Therefore, new and better methods are needed to increase the amount of available DNA and to quantitate detection of gene methylation status for several genes.